259 A RAPID, NON-PCR-BASED BOVINE EMBRYO BIOPSY SEXING ASSAY

2009 ◽  
Vol 21 (1) ◽  
pp. 227 ◽  
Author(s):  
R. Bleher ◽  
W. Erwin ◽  
A. M. Paprocki ◽  
C. M. Syverson ◽  
R. Koppang ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Blastocyst Stage ◽  
Bright Spot ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Gender Assignment ◽  
Specific Sequence ◽  
Laboratory Equipment

Sex determination of bovine embryos in conjunction with embryo transfer is an established method to maximize efficiency by producing offspring with the preferred gender. Present methodology for sexing embryos is based on PCR, which requires molecular laboratory equipment, trained personnel, and several hours before a result becomes known. We developed a simple, rapid, non-PCR procedure for identification of Y-chromosomes in bovine blastomeres recovered via biopsy. A biopsy (n = 5 to 8 blastomeres) was taken from a blastocyst-stage embryo and transferred onto a plastic slide. After drying and fixation, the cells were denatured and incubated with a peptide nucleic acid probe designed to target unique Y-chromosome specific sequence. The probe was conjugated to a fluorescent dye (CY-3) which enables Y-chromosome detection as a bright spot within blastomere nuclei when using a fluorescent microscope. The absence of signal indicates female embryonic DNA. From placement of the biopsy onto a plastic slide, the methodology required approximately 75 minutes to determine embryo gender. The accuracy of the biopsy sexing procedure was demonstrated by parallel gender determination of the same embryo using an established PCR method designed for the bovine amelogenin locus. Based on 18 in vitro-produced bovine embryos generating a result for both assays, there was a 94.4% match (17/18) of gender assignment. The present technology represents a simple alternative to PCR-based embryo sexing technology. Research is ongoing for future development of live embryo sexing determination.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

22 Vitrification of bovine embryo using antifreeze polyamino acid

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
T. Fujikawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Basal Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Polyamino Acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals 221 TYPICAL HIF1-REGULATED GENES ARE UNALTERED BY OXYGEN IN BOVINE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 261
Author(s):  
A. Harvey ◽  
K. Kind ◽  
J. Thompson
Keyword(s):  
Gene Expression ◽  
Oxygen Concentration ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Expression Of Genes ◽  
Regulated Gene Expression

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) post-compaction oxygen conditions moderate changes in gene expression are observed in the bovine blastocyst (Harvey et al. 2004 Biol. Reprod. 71, in press), compared with 3–4 fold increases in the mouse (Kind et al. 2004 Mol. Reprod. Dev., in press). Specifically, GLUT-1 (Harvey et al. 2004), myotrophin, and anaphase-promoting complex 1 (Harvey et al., unpublished) mRNAs are increased in bovine blastocysts following 2% oxygen culture, compared with those cultured under 20% oxygen. These oxygen-mediated differences in gene expression in the bovine are most likely regulated by hypoxia-inducible factor (HIF)2 transcription factor activity, as we have previously observed that HIF1α protein is not detectable in bovine embryos whereas HIF2α is readily detectable (Harvey et al. 2004). The aim of this study was to determine the effect of post-compaction oxygen concentration on the expression of typically HIF1-regulated and potential HIF2-regulated (suggested from a mouse knockout study; Scortegagna et al. 2003 Nat. Genet. 35, 371) genes in bovine blastocysts. In vitro-produced bovine embryos were generated using standard protocols. Compact morulae were randomly allocated to treatments under 2%, 7%, or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and samples were reverse-transcribed using Superscript II (Invitrogen, Melbourne, Australia). Amplification and analysis of cDNA was achieved by real-time PCR using specific primers and Sybr green PCR master mix (Applied BioSystems, Melbourne, Australia). Statistically significant differences in gene expression were analyzed by ANOVA, P < 0.05. Examination of expression of genes known to be regulated by HIF1 in somatic cells (reviewed by Semenza 2002 Biochem. Pharm. 64, 993) revealed no oxygen-mediated alteration in expression of aldose reductase, cyclooxygenase 2, or inducible nitric oxide synthase. However, the expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001). Expression of glutathione peroxidase, and CuZn- and Mn-superoxide dismutase (putative HIF2-regulated genes) was not influenced by oxygen concentration post-compaction. This study suggests that typical HIF1-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo. These results complement previous observations that HIF1α protein is not detectable in blastocyst-stage bovine embryos, and suggest that LDHA may be an HIF2 target gene in the bovine embryo. As embryo development is influenced by oxygen concentration, levels of LDHA at the blastocyst stage may be used as a marker of oxygen responsiveness.

scholarly journals Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems

Reproduction ◽  
2001 ◽  
pp. 601-610 ◽  
Author(s):  
MA Yaseen ◽  
C Wrenzycki ◽  
D Herrmann ◽  
JW Carnwath ◽  
H Niemann
Keyword(s):  
Growth Factor ◽  
Polyvinyl Alcohol ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Culture Systems ◽  
Igf I ◽  
Oviduct Fluid

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

Effect of α-tocopherol on in vitro culture of bovine embryos

2007 ◽  
Vol 87 (4) ◽  
pp. 539-542 ◽  
Author(s):  
A. Marques ◽  
G. Antunes ◽  
P. Santos ◽  
A. Chaveiro ◽  
F. Moreira da Silva
Keyword(s):  
Culture Media ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Media Control ◽  
Oviductal Fluid ◽  
Bovine Oocytes ◽  
Key Words Antioxidant

Bovine oocytes were matured and fertilised under 5% CO2. Presumptive zygotes were co-cultured in synthetic oviductal fluid droplets, supplemented with either 0 (control), 25, 50, 100, or 200 µM of α-tocopherol. Blastocyst development rates were significantly influenced (P < 0.05) by the level of antioxidant in the culture media. Control showed a blastocyst yield of 18.46, 21.11, 27.92, and 31.66%, respectively, at α25; α50 and α100. Blastocyst yield for α200 was severely decreased, to 8.01%. An increase in overall IVF results was also observed as the concentration of α-tocopherol increased (control, 14.21%; α25, 14.35%; α50, 19.52%; α100, 21.11%), decreasing to 5.75% for the concentration of α200. The present study clearly demonstrates that α-tocopherol at a concentration of 100 µM significantly improves the proportion of oocytes that develop to the blastocyst stage. Key words: Antioxidant, α-tocopherol, reactive oxygen species, bovine, embryo

scholarly journals Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

236 EFFECT OF HEAT STRESS DURING OOCYTE MATURATION ON GENE EXPRESSION OF IN VITRO-FERTILIZED AND PARTHENOGENETIC BOVINE BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 276
Author(s):  
F. Q. Costa ◽  
M. M. Pereira ◽  
S. Wohlres-Viana ◽  
R. V. Serapiao ◽  
B. C. Carvalho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Rna Extraction ◽  
Residual Effect ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Peroxiredoxin 1

Heat stress has been shown to have detrimental effects (41°C) during the first 12 h of in vitro maturation on bovine embryo development (Edwards JL and Hansen PJ 1996 Biol. Reprod. 55, 341-346). However, little is known about the effect on gene expression of in vitro-fertilized andparthenogenetic bovine embryos. This study evaluated the gene expression of in vitro-fertilized andparthenogenetic blastocysts derived fromheat-stressed oocytes. The transcripts evaluated were associated with genes encoding proteins involved in blastocoel formation [aquaporin (Aqp) 3 and Na+/K+-ATPase alpha 1; Watson AJ and Barcroft LC 2001 Frontiers Biosci. 6:d708-730] and cell viability (Bax and Peroxiredoxin 1; Van Delft MF and Huang DCS 2006 Cell Res. 16, 203-213; RaguS etal. 2007 PNAS104, 9747-9752). Oocytes were in vitro matured for 12 h at 41°C followed by 12 h at 38.5°C (heat-stressed oocytes; HS) or for 24 h at 38.5°C (non-heat-stressed oocytes; NHS) under 5% CO2. Heat-stressed and NHS oocytes were in vitro fertilized with Holstein sperm (HS-IVF and NHS-IVF subgroups, respectively) or activated with ionomycin and 6-DMAP (HS-PART and NHS-PART subgroups, respectively). Presumptive zygotes were cultured in CR2aa medium under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Embryos at blastocyst stage with same quality grade for all subgroups were obtained from 3 different replicates and distributed in pools of 10 embryos for relative quantification of the target transcripts. RNA extraction and reverse transcription were performed and cDNA quantified by real-time PCR. Transcripts of H2a gene were used as endogenous control, and statistical analysis was performed by pair-wise, fixed reallocation randomization test. Gene expression comparisons were performed between HS-IVF and NHS-IVF, HS-PART and NHS-PART, NHS-PART and NHS-IVF, and HS-PART and HS-IVF subgroups. Blastocyst rate is shown as mean ± SEM and relative expression as n-fold. The heat stress on oocytes during in vitro maturation decreased (P < 0.05, ANOVA) the development of presumptive zygotes to blastocyst stage at Day 8 for in vitro-fertilized (19.9 ± 2.9% and 10.5 ± 2.0% for NHS-IVF and HS-IVF, respectively) and parthenogenetic (33.0 ± 1.8% and 22.8 ± 2.8% for NHS-PART and HS-PART, respectively) embryos. Embryos from the HS-IVF subgroup showed less (P < 0.01) expression of Na+/K+-ATPase alpha 1 (0.67-fold) than did NHS-IVF embryos, whereas no difference was found for others genes. Embryos from the HS-PART subgroup showed less (P < 0.01) expression of Aqp 3 (0.77-fold) and greater (P < 0.05) expression of Bax (1.40-fold) than did NHS-PART embryos. Expression of Aqp 3 was up-regulated (P < 0.01) in NHS-PART (1.42-fold) embryos when compared with NHS-IVF ones, whereas expression of Na+/K+-ATPase alpha 1 (1.42-fold), Bax (1.67-fold), and Peroxiredoxin 1 (1.40-fold) were up-regulated (P < 0.05) in HS-PART embryos when compared with HS-IVF embryos. In conclusion, heat stress on oocytes during in vitro maturation can affect the amount of transcripts of in vitro-fertilized andparthenogenetic blastocysts, suggesting a residual effect on gene expression of bovine embryos. Financial support was provided by CNPq and FAPEMIG.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

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